CN109912830A - Dopamine/bacterial cellulose gel preparation method - Google Patents
Dopamine/bacterial cellulose gel preparation method Download PDFInfo
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- CN109912830A CN109912830A CN201910177835.7A CN201910177835A CN109912830A CN 109912830 A CN109912830 A CN 109912830A CN 201910177835 A CN201910177835 A CN 201910177835A CN 109912830 A CN109912830 A CN 109912830A
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- dopamine
- bacterial cellulose
- gel
- cellulose gel
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Abstract
The invention belongs to biomaterial preparation field more particularly to dopamine/bacterial cellulose gel preparation methods.Bacteria cellulose (BC) material has excellent mechanical property, such as high-intensitive, high-flexibility, high retentiveness etc..And it is non-degradable in vivo, but the poor ability of its induced tissue growth in vivo, and it is difficult to.In view of the above problems, bacteria cellulose is prepared into gel state by the preparation method of dopamine/bacterial cellulose gel provided by the invention, then modified by the surface of material, one layer of dopamine (PDA) film is coated, the induced tissue growth ability of gel is improved.
Description
Technical field
The invention belongs to biomaterial preparation field more particularly to dopamine/bacterial cellulose gel preparation methods.
Background technique
With the improvement of living standards, people increasingly focus on the pursuit for external beauty, injection cosmetology is because of operation
Risk is low, few intercurrent disease, and recovery time is short and by high praise.Injecting cosmetology is by injectable materials direct injection in human body
Privileged site makes the appearance of human body or the bodily form be taken on a new look, and plays the method promoted appearance beauty or improve function simultaneously.Injection beauty
Capacity materials are varied, have neuromuscular toxin, both creotoxin；Have soft tissue filling material, as collagen, sodium hyaluronate, from
Body cell etc.；The different material of the effects of there are also phosphatidyl choline, growth hormone, platelet rich plasmas mechanism.The above material exists
It can degrade in vivo, external longest is 17 months, and domestic longest is 8 months, to be injected again later, and such expense will cause
The pain and financial burden of patient.
Bacteria cellulose (BC) material has excellent mechanical property, such as high-intensitive, high-flexibility, high retentiveness etc..And
And it is non-degradable in vivo, but the poor ability of its induced tissue growth in vivo, and it is difficult to.In view of the above problems,
Bacteria cellulose is prepared into gel state by the present invention, then modified by the surface of material, coats one layer of dopamine (PDA)
Film improves the induced tissue growth ability of gel.
Summary of the invention
The present invention provides a kind of preparation method of dopamine/bacterial cellulose gel, comprising the following steps:
1) bacteria cellulose for taking 100g to be lyophilized, is crushed dry state bacteria cellulose with high speed Universal pulverizer.
2) NaOH, urea and water will be added in above-mentioned bacteria cellulose powder, is placed in refrigerator standing, formed transparent uniform solidifying
Gluey system.
3) by above-mentioned steps 2) obtain gel system, be centrifuged off bubble, obtain transparent bacterial cellulose solution.
4) epoxychloropropane (ECH) is added in above-mentioned bacterial cellulose solution as crosslinking agent, after stirring at room temperature,
Reaction obtains bacterial cellulose gel at 70 DEG C.
5) above-mentioned bacterial cellulose gel is dialysed in water.
6) take 0.5ml tir-HCl (TRIS buffer) that 50ml deionized water is added.
7) the above-mentioned system of 30ml (6) is taken, 50mg dopamine is added, 30s is pink.
8) step 7) obtain product in be put into the bacterial cellulose gel (4) dialyse, stirring, obtain dopamine/
Bacterial cellulose gel.
Dopamine/bacterial cellulose gel of preparation method preparation provided by the invention is non-degradable in vivo, can keep
Long-term filling effect.Compared with other similar patent, other patents are using (the dispersion after smashing of bacteria cellulose slurry
System), the present invention is to provide transparent uniform systems, it is ensured that dopamine is dispersed in gel.The present invention provides
Dopamine/bacterial cellulose gel and cell when co-culturing, cell viability is high.
Detailed description of the invention
Fig. 1 is the schematic diagram that dopamine and bacteria cellulose are crosslinked；
Fig. 2 is that dopamine/bacterial cellulose gel that one embodiment of the invention is prepared and cell co-culture copolymerization coke
Microphoto；
Fig. 3 is that dopamine/bacterial cellulose gel that one embodiment of the invention is prepared and cell co-culture at any time
The survival rate test result figure of growth.
Specific embodiment
Bacteria cellulose used in the embodiment of the present invention and Chinese patent, application No. is public in 201410089918.8
The bacteria cellulose opened is substantially identical, is by acetobacter xylinum (Acetobacter xylinum) metabolism in acetobacter
Product is made, and chemical component is that glucopyranose monomer (β-D-Glucose) is formed by β-Isosorbide-5-Nitrae-glucosides key connection
A kind of no branch, big molecule side chain polymer, be made of unique filamentary fibers, fibre diameter is each between 3~4nm
Filamentary fibers bandwidth is up to 30~100nm, and purity is up to reaching 99%, without hemicellulose, lignin, pectin and its
His cell wall components.
The cell and reagent information that specific embodiment is related to are as follows:
Rat fibroblast system (RFL-6) is purchased from Wei Si and rises biological medicine science and technology, and DMEM culture medium is purchased from U.S. Gibco
Company, trypsase are purchased from green skies company, China, and fetal calf serum is purchased from Gibco company, the U.S., and it is public that PBS is purchased from China fir in Beijing
Department, Pen .- Strep solution (100X) are purchased from green skies company, China, and DMSO is purchased from Amresco company, the U.S., CCK8 reagent
Box is purchased from Sigma Co., USA.
Embodiment one
One, long-acting type dopamine/bacterial cellulose gel preparation
The following steps are included:
1) bacteria cellulose for taking 100g to be lyophilized, is first crushed dry state bacteria cellulose with high speed Universal pulverizer, is crushed
Machine mixing speed is 8000rmp.
2) NaOH, urea and water will be added in above-mentioned bacteria cellulose powder, weight ratio BC:NaOH: urea: water=
1:10:12:77 is placed in -24 DEG C of refrigerators, 24 hours, forms transparent uniform gel system
3) by above-mentioned steps 2) the gel system that obtains, 20min, which is centrifuged, under -4 DEG C, 4000rpm revolving speed goes out bubble,
Obtain transparent bacterial cellulose solution.
4) 3ml epoxychloropropane (ECH) is added in above-mentioned bacterial cellulose solution as crosslinking agent, is stirred at room temperature
After 20min, 6h is reacted at 70 DEG C and obtains bacterial cellulose gel.
5) above-mentioned bacterial cellulose gel dialyses 4 days in ultrapure water, it is primary to change water within every 12 hours.
6) take 0.5ml tir-HCl (TRIS buffer) that 50ml deionized water is added.Survey ph=8.47.
7) the above-mentioned system of 30ml (6) is taken, 55mg dopamine is added, 30s is pink.
8) it is put into the bacterial cellulose gel (4) dialysed in the product that step 7) obtains, stirs 20min, obtains more
Bar amine/bacterial cellulose gel
Two, the case where dopamine/bacterial cellulose gel and cell that microexamination is prepared co-culture
(1) cell culture
RFL-6 cell culture is put in the DMEM culture medium containing penicillin and streptomycin (each 1U/mL, 0.1mg/mL) 10%FBS
It sets and is cultivated in 37 DEG C, 5%CO2, the cell incubator that relative humidity is 100%, cell adherent growth is used by secondary culture
In subsequent experimental.
(2) gel solution is prepared
Gel is released in the syringe 100ul extremely centrifuge tube containing 10mL culture medium and is dissolved, being configured to concentration is
1% mother liquor, when use, are diluted.
(3) cell and gel co-culture
(4) it dyes
(1) by the good RFL-6 cell inoculation of growth conditions in tissue culture plate, be placed in incubator culture (37 DEG C,
5%CO2).
(2) completely adherent to cell, routine culture is normally organized, experimental group is separately added into 0.05% gel rubber material effect
In cell 48h.
(3) cell is collected in digestion, and 105~106 cells are suspended in 1mL culture medium, 10 μ L Heochest are added
33342 dye liquors mix, 37 DEG C of incubation 5-15min.
(4) for cell in 4 DEG C, 500-1000r/min centrifugation 5min discards supernatant liquid.
(5) 1mL Buffer A working solution (10 × Buffer A is diluted 10 times with distilled water) suspension cell is added, adds
Enter 5 μ L PI dye liquors, is mixed after room temperature avoid light place 5-15min；
(6) confocal microscopy is taken pictures: the ultraviolet light that Heochest 33342 is excited with krypton laser, excitation wavelength are
352nm, launch wavelength are 400~500nm, generate blue-fluorescence；PI excites fluorescence using argon laser, and excitation light wave is a length of
488nm, wavelength of transmitted light are greater than 630nm, generate red fluorescence.
Picture that microscope is taken pictures as shown in Fig. 2,.
Three, survival experiment
(1) postdigestive cell is taken, counts adjustment cell concentration to 1 × 105/mL。
(2) it is inoculated in 96 orifice plates respectively, every 100 μ L of hole, every group of cell sets 3 multiple holes.After cell is completely adherent just
Normal groups of cells routine culture, experimental group are separately added into 0.05% gel.
(3) will 96 orifice plates move into incubator in cultivate (37 DEG C, 5%CO2), culture for 24 hours, after 48h, 72h carry out CCK-8 inspection
It surveys.
(4) 10 μ L CCK-8 solution (being careful not to generate bubble) is added in every hole, in being incubated for 1-4h in incubator.
(5) absorbance value at 450nm is measured with microplate reader.
(6) it is arranged blank well (culture medium, CCK) simultaneously, control wells (untreated cell, culture medium, CCK).
Cell viability (%)=[A (experimental group)-A (blank group)]/[A (control group)-A (blank group)] × 100%
Cell inhibitory rate (%)=[A (control group)-A (experimental group)]/A (control group)=1- cell viability
A (experimental group): the absorbance value with treated cell, CCK solution
A (blank group): have culture medium and CCK solution without the absorbance value in the hole of cell
A (control group): the absorbance value with untreated cell, CCK solution.
Test results are shown in figure 3, illustrates dopamine provided by the invention/bacterial cellulose gel induced cell growth
Ability is fine.
Claims (8)
1. a kind of preparation method of dopamine/bacterial cellulose gel, which comprises the following steps:
1) bacteria cellulose for taking 100g to be lyophilized, is crushed dry state bacteria cellulose with high speed Universal pulverizer.
2) NaOH, urea and water will be added in above-mentioned bacteria cellulose powder, is placed in refrigerator standing, forms transparent uniform gel
System.
3) by above-mentioned steps 2) obtain gel system, be centrifuged off bubble, obtain transparent bacterial cellulose solution.
4) epoxychloropropane (ECH) is added in above-mentioned bacterial cellulose solution as crosslinking agent, after stirring at room temperature, 70
Reaction obtains bacterial cellulose gel at DEG C.
5) above-mentioned bacterial cellulose gel is dialysed in water.
6) take 0.5ml tir-HCl (TRIS buffer) that 50ml deionized water is added.
7) the above-mentioned system of 30ml (6) is taken, 50mg dopamine is added, 30s is pink.
8) it is put into the bacterial cellulose gel (4) dialysed in the product that step 7) obtains, stirs, obtains dopamine/bacterium
Cellulose gel.
2. a kind of preparation method of dopamine/bacterial cellulose gel as described in claim 1, which is characterized in that the step
It is rapid 2) in bacteria cellulose: NaOH: urea: water (weight ratio)=1:10:12:77, refrigerator temperature are set as -24 DEG C, when standing
Between be 24 hours.
3. a kind of preparation method of dopamine/bacterial cellulose gel as claimed in claim 1 or 2, which is characterized in that described
Centrifugal rotational speed is 4000rpm in step 3), is centrifuged 20min at -4 DEG C.
4. a kind of preparation method of dopamine/bacterial cellulose gel as claimed in claim 1 or 2, which is characterized in that described
The dosage of epoxychloropropane is 3ml, reaction time 6h in step 4).
5. a kind of preparation method of dopamine/bacterial cellulose gel as claimed in claim 3, which is characterized in that the step
It is rapid 4) in epoxychloropropane dosage be 3ml, reaction time 6h.
6. a kind of preparation method of dopamine/bacterial cellulose gel as claimed in claim 1 or 2, which is characterized in that described
Dialysis in step 5) carries out in ultrapure water, and dialysis time is 4 days, and it is primary to change water within every 12 hours.
7. a kind of preparation method of dopamine/bacterial cellulose gel as claimed in claim 3, which is characterized in that the step
It is rapid 5) in dialysis carried out in ultrapure water, dialysis time be 4 days, it is primary to change water within every 12 hours.
8. a kind of preparation method of dopamine/bacterial cellulose gel as claimed in claim 4, which is characterized in that the step
It is rapid 5) in dialysis carried out in ultrapure water, dialysis time be 4 days, it is primary to change water within every 12 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115721767A (en) * | 2022-12-12 | 2023-03-03 | 广东省人民医院 | Antibacterial healing-promoting porous medical dressing and preparation method thereof |
| CN116942915A (en) * | 2023-08-09 | 2023-10-27 | 山东大学齐鲁医院 | Bone cell gel culture method and application thereof in bone echinococcosis bone defect |
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| CN101445609A (en) * | 2008-11-14 | 2009-06-03 | 武汉大学 | Hydroscopic cellulose hydrogel and preparation method thereof |
| CN103861146A (en) * | 2014-03-13 | 2014-06-18 | 海南光宇生物科技有限公司 | Bacterial cellulose biological patch and manufacturing method thereof |
| CN106076283A (en) * | 2016-06-11 | 2016-11-09 | 华南理工大学 | A kind of nano-cellulose/poly-dopamine hydrogel adsorbent and preparation method and application |
| CN108359056A (en) * | 2018-03-06 | 2018-08-03 | 中国科学院理化技术研究所 | A kind of self-healing hydrogel and its preparation method and application of cellulose-dopamine-polymer composites |
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Patent Citations (4)
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|---|---|---|---|---|
| CN101445609A (en) * | 2008-11-14 | 2009-06-03 | 武汉大学 | Hydroscopic cellulose hydrogel and preparation method thereof |
| CN103861146A (en) * | 2014-03-13 | 2014-06-18 | 海南光宇生物科技有限公司 | Bacterial cellulose biological patch and manufacturing method thereof |
| CN106076283A (en) * | 2016-06-11 | 2016-11-09 | 华南理工大学 | A kind of nano-cellulose/poly-dopamine hydrogel adsorbent and preparation method and application |
| CN108359056A (en) * | 2018-03-06 | 2018-08-03 | 中国科学院理化技术研究所 | A kind of self-healing hydrogel and its preparation method and application of cellulose-dopamine-polymer composites |
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| CHUNYU CHANG ET AL.: ""Structure and properties of hydrogels prepared from cellulose in NaOH/urea aqueous solutions"", 《CARBOHYDRATE POLYMERS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115721767A (en) * | 2022-12-12 | 2023-03-03 | 广东省人民医院 | Antibacterial healing-promoting porous medical dressing and preparation method thereof |
| CN115721767B (en) * | 2022-12-12 | 2024-03-12 | 广东省人民医院 | Antibacterial healing-promoting porous medical dressing and preparation method thereof |
| CN116942915A (en) * | 2023-08-09 | 2023-10-27 | 山东大学齐鲁医院 | Bone cell gel culture method and application thereof in bone echinococcosis bone defect |
| CN116942915B (en) * | 2023-08-09 | 2024-02-13 | 山东大学齐鲁医院 | Bone cell gel culture method and application thereof in bone echinococcosis bone defect |
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